Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Production and characterization of monoclonal antibodies to anti-human chorionic somatomammotropin by immunization with two free synthetic peptides

The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.     

Monoclonal antibodies specific for human polymorphic cell surface antigens. I. Evaluation of methodologies. Report on a workshop

Fire techniques, the direct and the antiglobulin enhanced cytotoxicity assays, indirect immunofluorescence, the enzyme-linked immunosorbent assay, and the radioimmunoassay, were evaluated in a workshop to determine their utility in studies of the interactions of monoclonal antibodies with HLA antigens expressed on lymphocytes. Several well-defined antibodies, both cytotoxic and noncytotoxic, were tested against well-characterized human lymphoid cells. All the methods suffer from some deficiency. The enhanced cytotoxicity assay, however, is most useful as a routine screening tool because of its ease and simplicity: whereas, the enzyme-linked immunosorbent assay is most useful when dissection of antigenic structure is sought because it yields information on the quantities of the antigenic determinants expressed on the cell surface without requiring radioactive reagents.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

Production and Characterization of Monoclonal Antibodies against Bovine Luteinizing Hormone

Five mouse hybridoma cell lines producing monoclonal antibody against bovine luteinizing hormone (LH) have been established and the respective antibodies characterized by radioimmunoassay, immunofluorescence and immunoelectrophoresis. All antibodies belong to the IgG class and bind to staphylococcus protein A. Intraspecies cross-reactivity studies revealed no reaction with bovine follicle stimulating hormone (FSH). However, all antibodies showed partial cross-reaction with bovine thyroid stimulating hormone (TSH) suggesting a close conformational similarity between bovine LH and TSH. Studies on interspecies cross-reactivity (rat and human) showed that three of these five antibodies strongly react with rat LH but not at all with either rat FSH or rat TSH thus representing monospecific reagents for investigations concerning LH in this species. One of these three antibodies also strongly binds to human LH and to the same extent to human chorionic gonadotropin (CG) but not to human FSH or TSH. It was concluded that at least three different epitopes on the bovine LH molecule are recognized and that they are located on the β-chain of the hormone.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.     

Differences in the expression and release of DR, BR, and DQ molecules in human cells treated with recombinant interferon gamma: comparison to other interferons

This study presents a comparative analysis of the effects of different interferons (IFN) on the three recognizable subsets of human HLA class II molecules: DR, BR, and DQ. Both cellular expression and shedding of class II molecules have been determined on three different cell types. The results can be summarized as follows: class II molecules are markedly increased by IFN gamma; IFN beta has a lower enhancing effect, and IFN alpha has only a slight, if any, effect. Kinetically, the action of IFN gamma is prompter and longer lasting than that of IFN beta. DQ expression is much more enhanced by IFN gamma than either DR or BR; IFN beta has the same effect on all three subsets. Parallel changes of the cellular expression and of the shedding of these molecules are observed. A melanoma and a lymphoblastoid cell line and peripheral blood mononuclear cells show qualitatively similar modifications.